The objective of this proposal is to investigate the roles of membrane factors in amyloid-p peptide (A(3)- induced oxidative stress and inflammatory responses in glial cells. Here membrane factors include local membrane phase properties and cytoskeletal linkage of membrane receptors. Ap-induced oxidative stress and inflammation are implicated in Alzheimer's disease (AD). In fact, Ap has been found to induce oxidative stress through activation of NADPH oxidase. Excess reactive oxygen species (ROS) in cells, in turn, cause oxidative damage including lipid peroxidation, oxidation of RNA, DMA, and proteins, which subsequently perturb normal cellular processes, including intracellular signaling and cytoskeleton organization. In this regard, we have previously reported that oxidative stress causes astrocyte membrane to become more gel- like through activations of p38 MARK and of cytosolic phospholipase A2 (cPLA2). Our preliminary results also show Ap42 oligomers induce activation of cPLA2. Since it has been reported that the efficiency of NADPH oxidase activation is dependent of its local membrane environment and our preliminary studies show that the membrane subunit of NADPH oxidase, gp91ph0*, is predominately localized at the high GP domains (i.e. more gel-like membranes) in astrocytes, these findings lead us to hypothesize that Af!*? induces cPLA? activation through activations of NADPH oxidase and MAPK pathways to cause glial membranes to become more gel-like, which, in turn, becomes a positive feedback to further amplify the activation of NADPH oxidase to produce ROS. Other membrane factors, such as cytoskeletal linkages of membrane receptors, can also be a fundamental element governing cell functions. It has been reported that Ap42 binds to membrane receptors, CD36, ctePi, CD47 and scavenger receptor class A, resulting in inflammatory responses in microglial cells, which can be suppressed by blocking the binding of Ap42 to one of these receptors using their antibodies. These findings lead us to hypothesize that cooperativitv between these membrane receptors is required for AB-induced inflammatory responses in microglial cells and this coooerativitv is established through the cvtoskeletal linkages of these membrane receptors. Since oxidative stress and inflammation are implicated in AD, our study on how membrane factors involved in the mechanisms of Ap-induced oxidative stress and inflammatory responses in glial cells will prove critical to deepen our understandings in the pathogenesis of AD. Novel biophysical techniques including fluorescence imaged deformation (FIMD) and fluorescent microscopy of LAURDAN, and various biochemical techniques will be applied to accomplish this proposed project. University of Missouri-Columbia, Columbia, MO PHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Lee, James, Chak-Man KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Lee, James leejam University of Missouri-Columbia Sun, Grace SUNGRACE University of Missouri-Columbia Haidekker, Mark mhaidekk University of Missouri-Columbia in the format shown below. Role on Project Principal Investigator Co-Principal Investigator Co-Investigator OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Human Embryonic Stem Cells E3 No l~l Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/reqistrv/index.asp. Usecontinuation pages as needed. If a specific line cannot be referenced at this lime, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. I Yes DNO PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Lee, James, Chak-Man The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,